TW202246518A - Genetically modified oncolytic herpes simplex virus delivering chemokine and tumor associated/specific antigen - Google Patents

Abstract

Disclosed is a genetically modified oncolytic herpes simplex virus (oHSV) encoding a truncated nonsignaling variant of at least one tumor associated/specific antigen, and at least one chemokine. The expression of the truncated nonsignaling variant and the chemokine is under the control of an immediate-early gene promoter of HSV, and the truncated nonsignaling variant is expressed and presented on a tumor cell surface as a biomarker upon replication of the oHSV in the tumor cell, and the chemokine is expressed and released to induce chemotaxis of an immune cell towards the tumor cell. The genetically modified oHSV can be used in combination with CAR-T, ADC, and/or BiTE therapies.

Description

Genetically Modified Oncolytic Herpes Simplex Diseases Delivering Chemokines and Tumor-Associated/Specific Antigens poison

The present invention relates to an oncolytic herpes simplex virus genetically engineered to carry a gene encoding at least one chemokine and/or at least one tumor-associated/specific antigen, and its combination with tumor-targeting therapeutic agents, including CAR -T, BiTE and ADC) combination for the treatment of various tumor applications.

Adoptive transfer of CAR-T cells in patients with hematologic malignancies has shown striking results. However, this approach has had little effect in patients with solid tumors. It seems unlikely that CAR-T cell therapy alone will be sufficient to induce a complete response in most cancers. Combining CAR-T cells with other cancer therapies that have different mechanisms of action and have the potential to synergize with T cells may reduce tumor escape and increase the success rate of CAR-T cell therapy.

Oncolytic virotherapy is a treatment for cancer that uses natural or genetically modified viruses that selectively replicate within cancer cells. The field of oncolytic virotherapy has received renewed attention following FDA approval of Talimogene laherparepvec (T-VEC), an oncolytic herpes simplex virus type 1 (HSV-1) modified to express GM-CSF. In addition, oncolytic virus (OV) can be further modified to selectively deliver therapeutic transgenes to the tumor microenvironment to enhance its antitumor efficacy or enhance antitumor immune response. Preclinical studies combining CAR-T cells with oncolytic viruses carrying cytokines, chemokines, BiTEs, or immune checkpoint inhibitors resulted in enhanced therapeutic efficacy. For example, oncolytic adenoviruses modified to express IL-15 and RANTES or IL-2 and TNF-α have been shown to increase the accumulation and survival of CAR-T cells in the tumor microenvironment. Vaccinia virus expressing CXCL11, a CXCR3 ligand, was used to attract effector cells after transfer to enhance intratumoral trafficking of CAR-T cells. Another study showed that expression of oncolytic adenoviruses via BiTEs targeting a second tumor antigen could resolve the heterogeneity of antigen expression. As expected, all these combinations of CAR-T cells and armed OVs enhanced tumor control and prolonged survival compared with each agent as monotherapy.

A recent study engineered oncolytic viruses to express a non-signaling, truncated CD19 (CD19t) protein for tumor-selective delivery, enabling targeting by CD19-CAR T cells. Infection of tumor cells with an oncolytic vaccinia virus encoding CD19t (OV19t) produces de novo synthesis of CD19 on the cell surface prior to virus-mediated tumor lysis. Co-cultured CD19-CAR T cells secreted cytokines and exhibited potent cytolytic activity against infected tumors. Using several mouse tumor models, delivery of OV19t promoted tumor control after CD19-CAR T cell administration. OV19t induces local immunity characterized by tumor infiltration of endogenous and adoptively transferred T cells. CAR T cell-mediated tumor killing also induces virus release from dying tumor cells, which promotes tumor expression of CD19t. Although the study cured more than 50% of the mice treated with this combination therapy, some mice had transient responses and some did not (Park et al., Sci.Transl.Med.12, eaaz1863(2020 )).

US20190233536A1 discloses a modified adenovirus, in particular Enadenotucirev (EnAd), armed with at least two bispecific T cell engagers (BiTEs), each comprising at least two binding domains, at least one of which Specific for a surface antigen on an immune cell of interest, such as a T cell of interest. Arming adenoviruses with BiTE molecules allows bispecific antibody fragment molecules to "piggyback" the ability of adenoviruses to selectively infect cancer cells, enabling targeted delivery of BiTEs to tumor cells. Once infected by adenoviruses, BiTE molecules are synthesized by tumor cells, secreted, and act locally, thereby spreading beyond the immediate area of viral coverage. Thus, this allows the BiTE to spread beyond the immediate site of infection, but at the same time restricts the spread of the virus too far beyond the infected tumor cell nest. This minimizes the risk of unwanted off-target effects.

definition

It is to be noted that the term "a" or "an" or "an" entity refers to one or more or one or more of that entity; for example, "truncated non-signaling variant" is understood to mean One or more truncated non-signaling variants. Accordingly, the terms "a" or "an", "one or more" or "one or more" and "at least one" or "at least one" may be used interchangeably herein.

As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, eg, F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, and the like. Regardless of structure, antibody fragments bind to the same antigens that are recognized by intact antibodies. The term "antibody fragment" includes aptamers, aptamer spiegelmers and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

Antibodies, antigen-binding polypeptides or variants or derivatives thereof of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, Epitope binding fragments (e.g. Fab, Fab' and F(ab') ₂ , Fd, Fvs, single chain Fvs (scFv), single chain antibody, disulfide-linked Fvs (sdFv)), comprising a VK or VH domain , fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies directed against the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecule of the present disclosure may be of any class (eg, IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2) of an immunoglobulin molecule. For example, an anti-PD-1 antibody may refer to an antigen-binding fragment thereof, such as a Fab fragment or scFv thereof.

"Specifically binds", "specifically" or "with specificity" generally means that an antibody binds an epitope via its antigen-binding domain, and that binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" an epitope when it binds to the epitope more readily through its antigen-binding domain than to a random, unrelated epitope. The term "specificity" is used herein to quantify the relative affinity with which an antibody binds to a certain epitope. For example, antibody "A" can be said to have a higher specificity for a given epitope than antibody "B", or antibody "A" can be said to bind to epitope "C" more than it does to a related epitope "D". ” is more specific.

As used herein, "cancer" or "tumor", as used interchangeably herein, refers to a group of diseases that are treatable in accordance with the present disclosure and involve the growth of abnormal cells, which may invade or spread to other parts of the body. Not all tumors are cancerous; benign tumors do not spread to other parts of the body. Possible signs and symptoms include: new lumps, unusual bleeding, prolonged cough, unexplained weight loss and changes in bowel movements, among others. There are more than 100 different known cancers affecting humans. As used herein, "cancer" includes, but is not limited to, solid cancers (eg, tumors) and hematological malignancies. "Hematological malignancies", also known as blood cancers, are cancers that originate in blood-forming tissues such as the bone marrow or other cells of the immune system. Hematologic malignancies include, but are not limited to, leukemias (e.g., acute myeloid leukemia (AML), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL ), hairy cell leukemia, and large granular lymphocytic leukemia), myelodysplastic syndrome (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocythemia, primary myelofibrosis, and chronic myeloid leukemia ), lymphoma, multiple myeloma, MGUS and similar disorders, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma Lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies. "Solid cancer" includes, but is not limited to, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, prostate cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, Uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophagus cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, Penile cancer, childhood tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumor, brainstem glioma, pituitary adenoma , Kaposi's sarcoma, epidermoid carcinoma, squamous Cell carcinoma, Environmentally induced cancers (including cancers induced by asbestos).

As used herein, the term "treatment" or "treatment" refers to both therapeutic treatment and prophylactic measures aimed at preventing or slowing down (lessening) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, lessening of disease extent, stabilization (i.e., not worsening) of disease state, delay or slowing of disease progression, amelioration or palliation of disease state, and resolution of symptoms (whether partial or total) ), whether detectable or undetectable. "Treatment" also means prolonging survival compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or disorder as well as those prone to have the disease or disorder or those in whom the disease or disorder is to be prevented.

"Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, livestock, farm and zoo animals, sport animals or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like.

As used herein, phrases such as "patient in need of treatment" or "subject in need of treatment" include subjects who benefit from administration of oHSV-1 or compositions of the present invention for, e.g., detection, diagnostic procedures, and/or treatment , such as a mammal object.

Those of ordinary skill in the art will also appreciate that modified genomes as disclosed herein may be modified such that they differ in nucleotide sequence from the modified polynucleotides from which they are derived. For example, a polynucleotide or nucleotide sequence derived from a given DNA sequence may be similar, such as having a certain percentage identity to the starting sequence, for example it may be 60%, 70%, 75% identical to the starting sequence , 80%, 85%, 90%, 95%, 98% or 99% of the same sex.

In addition, nucleotide or amino acid substitutions, deletions or insertions may be made to make conservative substitutions or changes in regions of "non-essential" amino acids. For example, a polypeptide or amino acid sequence derived from a given protein, except for one or more individual amino acid substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more single amino acid substitutions, insertions or deletions), the remainder may be identical to the starting sequence. In certain embodiments, the polypeptide or amino acid sequence derived from a given protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 individual amino acid substitutions relative to the starting sequence, insertion or deletion.

A "therapeutically effective amount" or "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improving a patient's health status, reducing tumor burden, arresting or slowing tumor growth, and/or non-metastasis of cancer cells to other parts of the body.

CAR-T cells are T cells expressing chimeric antigen receptors. T cells expressing CAR molecules may be helper T cells, cytotoxic T cells, virus-specific cytotoxic T cells, memory T cells, or γδ T cells. A chimeric antigen receptor (CAR) is a recombinant fusion protein comprising: (1) an extracellular ligand-binding domain, known as an antigen recognition domain, (2) a transmembrane domain, and (3) a signal transduction domain area. An extracellular ligand binding domain is an oligopeptide or polypeptide capable of binding a ligand. Preferably, the extracellular ligand binding domain is capable of interacting with cell surface molecules, which may be antigens, receptors, peptide ligands, protein ligands of targets or target peptides. In the present disclosure, the extracellular ligand binding domain will be capable of interacting with a truncated non-signaling variant of a tumor-associated antigen or a tumor-specific antigen.

Typically, the extracellular ligand-binding domain is linked to the signaling domain of a chimeric antigen receptor (CAR) through the transmembrane domain I. The transmembrane domain crosses the cell membrane, anchors the CAR on the surface of T cells, connects the extracellular ligand-binding domain to the signal transduction domain, and affects the expression of CAR on the surface of T cells. The transmembrane domain may further comprise a hinge region between the extracellular ligand binding domain and said transmembrane domain. The term "hinge region" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular ligand binding domain. In particular, the hinge region was used to provide more flexibility and accessibility to the extracellular ligand-binding domain. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids, more preferably 25 to 50 amino acids. The hinge region can be derived from naturally occurring molecules such as CD28, 4-1BB (CD137), OX-40 (CD134), CD3ζ, T cell receptor alpha or beta chain, CD45, CD4, CD5, CD8, CD8α, CD9, CD16 , CD22, CD33, CD37, CD64, CD80, CD86, ICOS, all or part of CD154, or derived from all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or the hinge region may be a fully synthetic hinge sequence.

Chimeric antigen receptors (CARs) also contain the signal transduction domain or intracellular signaling domain of CAR, which is responsible for intracellular signaling after the extracellular ligand-binding domain binds to the target, resulting in the activation of immune cells. Initiation and immune response. That is, the signaling domain is responsible for the initiation of at least one normal effector function of CAR-expressing immune cells. For example, the effector function of T cells can be cytolytic activity or helper T cell activity, including cytokine child secretion. Thus, the term "signal transduction domain" refers to the portion of a protein that transduces the effector signal and directs the cell to perform a specific function. Examples of signaling domains for CARs may be the cytoplasmic sequences of T cell receptors and co-receptors that cooperate to initiate signal transduction following antigen receptor engagement, and any derivatives or variants of these sequences and any synthetic sequence with the same functional capability. The signal transduction domain contains two distinct classes of cytoplasmic signaling sequences, those that initiate antigen-dependent primary initiation, and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals. Primary cytoplasmic signaling sequences may contain signaling motifs known as immunoreceptor tyrosine-based initiation motifs of ITAMs. ITAMs are well-defined signaling motifs present in the intracytoplasmic tails of multiple receptors that serve as binding sites for Syk/Zap70-like tyrosine kinases. Non-limiting examples of ITAMs that may be used in the present disclosure may include those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d. In one embodiment, the signaling domain of the CAR may comprise a CD3zeta signaling domain with an amino acid sequence having at least 80%, 90%, 95%, 97% or 99% sequence identity thereto. The present disclosure contemplates the use of the genetically engineered oHSV described herein in combination with any CAR-T, but is not limited thereto.

Typical antibody-drug conjugates (ADCs) contain monoclonal antibodies that bind to specific antigens on the surface of cancer cells. These antibodies include proteins on the surface of the immune system's B and T cells, such as CD20, CD22, and human epidermal growth factor receptor 2 (Her2) and prostate-specific membrane antigen (PSMA). These antibodies are linked to highly toxic drugs via a cleavable linker unit. Drugs are designed to induce irreversible DNA damage or interfere with cell division, leading to apoptosis in cancer cells. ADCs contain specific antigens that bind to the surface of cancer cells monoclonal antibody. These antibodies include proteins on the surface of the immune system's B and T cells, such as CD20, CD22, and human epidermal growth factor receptor 2 (Her2) and prostate-specific membrane antigen (PSMA). These antibodies are linked to highly toxic drugs via a cleavable linker unit. Drugs are designed to induce irreversible DNA damage or interfere with cell division, leading to apoptosis in cancer cells.

The mechanism of antibody-drug conjugates (ADCs) is to recognize and bind specific antigens through antibodies, trigger a series of reactions, and then enter the cytoplasm through endocytosis, where highly toxic drugs are dissociated from lysosomal enzymes to kill cancer cells. Compared with traditional chemotherapy, which indiscriminately causes damage to cancer cells and normal tissues, targeted drug delivery allows drugs to directly act on cancer cells and reduce damage to normal cells. A typical antibody-drug conjugate consists of three parts: drug, linker unit and antibody. The choice of specific antibody and drug depends on the specific disease and has an important impact on the safety and efficacy of the conjugate. The stability of the linker unit and the way of coupling with the antibody play a decisive role in the development of ADC drugs. Factors that determine the efficacy of antibody-drug conjugates include the stability and cleavage sensitivity of the linker unit, cell surface-triggered internalization, transport, and release of cytotoxins. This disclosure contemplates the use of the T7 series oHSVs described herein in combination with any ADC, but is not limited thereto.

Bispecific T cell engagers (BiTEs) are relatively simple bispecific molecules that are specific for the CD3E subunit of the TCR complex of T cells and also target an antigen of interest, such as a cancer antigen. Because BiTEs are specific for TCR complexes, this enables BiTEs to prime resident T cells to kill cells expressing specific target antigens on their cell surface, such as cancer cells. An important property of BiTEs is their ability to make CD4+ and unprimed CD8+ T The cells target cancer cells. That is, BiTE-primed T cells can kill cells independently of the MHC expression on the cell surface. This is important because some tumor cells downregulate MHC, making them resistant to drugs such as CAR-T cells and immTACs. Unfortunately, the cycling kinetics of BiTEs are poor compared to full-length antibodies. This means that when administered to a patient, most of the BiTEs do not reach their target cells. Furthermore, the use of high-affinity anti-CD3 ScFv as part of the BiTE resulted in robust binding to T cells in the blood, which also interfered with delivery to tumors. Therefore, BiTEs cannot realize their full potential as anticancer therapies because they cannot be efficiently delivered to tumor cells. This disclosure contemplates the use of oHSVs described herein in combination with any BiTE, but is not limited thereto.

As used herein, the term "tumor-associated/specific antigen" refers to a tumor-associated antigen, a tumor-specific antigen, or both. For example, the term "at least one tumor-associated/specific antigen" refers to at least one tumor-associated antigen or at least one tumor-specific antigen, and may include a pair of tumor-associated and tumor-specific antigens. For example, the term "truncated non-signaling variant of at least one tumor-associated/specific antigen" is intended to include truncated non-signaling variants of one tumor-associated antigen, two or more tumor-associated antigens, Truncated non-signaling variants of related antigens, truncated non-signaling variants of one tumor-specific antigen, truncated non-signaling variants of two or more tumor-specific antigens , truncated non-signaling variants of one tumor-associated antigen and two or more tumor-specific antigens, and truncated non-signaling variants of two or more tumor-associated antigens and one tumor-specific antigen Variants of signaling. For example, the term "two tumor-associated/specific antigens" may include two tumor-associated antigens, two tumor-specific antigens as well as one tumor-associated antigen and one Combinations of tumor-specific antigens.

As used herein, a "biomarker", "truncated non-signaling variant", "truncated variant" or "non-signaling variant" of a particular tumor-associated antigen or tumor-specific antigen A variant" refers to a variant of a tumor-associated antigen or a tumor-specific antigen that has been mutated, deleted, or otherwise modified to shut down signal transduction of its wild-type counterpart in a signaling pathway. The variant exposes at least some epitopes of the antigen such that the variant is capable of being bound by an antibody or antigen-binding fragment thereof (eg scFv) specific for the wild-type antigen. In cases where the antigen is a transmembrane protein, non-signaling variants of tumor-associated antigens or tumor-specific antigens are generally known to be the extracellular domain of the antigen, the extracellular-transmembrane domain of the antigen, or the The ectodomain or extracellular-transmembrane domain has at least 90% amino acid sequence identity and its equivalent. Equivalents are unable to transduce a signal, but expose at least some epitopes on the extracellular domain of the antigen.

For example, a non-signaling variant of CD19 (also referred to herein as non-signaling CD19) is the 323 amino acid extracellular-transmembrane domain of wild-type CD19 (SEQ ID NO: 14). Non-signaling BCMA is the 77 amino acid extracellular-transmembrane domain of wild-type BCMA (SEQ ID NO: 15). Non-signaling HER2 is the 675 amino acid extracellular-transmembrane domain of wild-type HER2 (SEQ ID NO: 16). Non-signaling Trop-2 is the 297 amino acid extracellular-transmembrane domain of wild-type Trop-2 (SEQ ID NO: 17).

Extracellular domains and transmembrane domains can be obtained without difficulty by routine practice in the art. The amino acid sequences of the extracellular/transmembrane domains of various tumor-associated antigens or tumor-specific antigens can be obtained from public sources including NCBI (https://www.ncbi.nlm.nih.gov/protein) . It should be pointed out that once the non-signaling variants are expressed on the tumor cell surface On the surface, it will be recognized and bound by antibodies or antigen-binding fragments of antibodies specific for tumor-associated antigens or tumor-specific antigens. The antigen-binding fragment can be part of a CAR-immune cell (eg, CAR-T cell or CAR-NK cell) or a BiTE. Antibodies can be conjugated with chemotherapeutic drugs to form ADCs. However, the non-signaling variants did not trigger signaling pathways like their wild-type counterparts. Those skilled in the art will readily test and verify whether a variant of a tumor-associated antigen or a tumor-specific antigen is non-signaling. For example, this can be determined by detecting the extent of downstream proteins in the known normal signaling pathway of the wild-type antigen.

Genetically Modified Oncolytic Herpes Simplex Virus (oHSV)

The present disclosure provides a genetically modified oncolytic herpes simplex virus (oHSV). A genetically modified oHSV is modified such that it exhibits a truncated non-signaling transducer of at least one tumor-associated or tumor-specific antigen when replicating in susceptible cells (eg, solid tumor cells). The present inventors demonstrated the successful expression of different truncated tumor-associated antigens or tumor-specific antigens on the cell surface following infection and replication of genetically modified oHSV in tumor cells. Non-signaling tumor-associated or tumor-specific antigens expressed and then presented on the surface of tumor cells mark the tumor cells as targets for various antigen-directed therapies, such as CAR-T therapies. In some embodiments, the genetically modified oHSV of the present disclosure is further modified such that it expresses at least one chemokine when replicating in susceptible cells (eg, solid tumor cells). The inventors have shown that secreted chemokines can be detected as early as 4 hours after infection and are retained for at least 4 days after infection with oHSV virus. Chemokine presentation and release induces chemotaxis of immune cells (e.g., T cells or CAR T cells) towards susceptible cells, which facilitates immune cell trafficking and infiltration into the tumor mass.

In some embodiments, there is provided a genetically modified oncolytic herpes simplex virus (oHSV), wherein a polynucleotide is incorporated into the genome of said oHSV, said polynucleotide encoding at least one tumor-associated/specific A truncated non-signaling variant of a sex antigen, wherein the expression of the truncated non-signaling variant is controlled by the immediate early gene promoter of HSV.

In some embodiments, there is provided a genetically modified oncolytic herpes simplex virus (oHSV), wherein a polynucleotide is incorporated into the genome of said oHSV, said polynucleotide encoding (a) at least one tumor-associated The truncated non-signaling variant of the sex/specific antigen, and (b) at least one chemokine, wherein the expression of the truncated non-signaling variant and the at least one chemokine is affected by the immediate expression of HSV Control of early gene promoters.

In some embodiments, there is provided a genetically modified oncolytic herpes simplex virus (oHSV), wherein a polynucleotide encoding (a) a tumor associated The truncated non-signaling variant of the antigen or a tumor-specific antigen, and (b) a chemokine, wherein the expression of the truncated non-signaling variant and the chemokine is controlled by HSV Control of immediate early gene promoters.

In some embodiments, there is provided a genetically modified oncolytic herpes simplex virus (oHSV), wherein a polynucleotide encoding (a) two tumor-associated A truncated non-signaling variant of a sex/specific antigen, and (b) a chemokine, wherein the expression of the truncated non-signaling variant and the chemokine is affected by the immediate early stage of HSV Control of gene promoters. In some embodiments, the two tumor-associated/specific antigens comprise two identical or different tumor-associated antigens. in some real In an embodiment, the two tumor-associated/specific antigens comprise two identical or different tumor-specific antigens. In some embodiments, the two tumor-associated/specific antigens comprise one tumor-associated antigen and one tumor-specific antigen.

In some embodiments, there is provided a genetically modified oncolytic herpes simplex virus (oHSV), wherein a polynucleotide is incorporated into the genome of said oHSV, said polynucleotide encoding (a) at least one tumor-associated A truncated non-signaling variant of a sex/specific antigen, and (b) two chemokines, wherein the expression of the truncated non-signaling variant and the chemokines is affected by the immediate expression of HSV Control of early gene promoters. In some embodiments, the two chemokines are the same. In some embodiments, the two chemokines are different.

In some embodiments, there is provided a genetically modified oncolytic herpes simplex virus (oHSV), wherein a polynucleotide encoding (a) two tumor-associated A truncated non-signaling variant of a sex/specific antigen, and (b) two chemokines, wherein the expression of the truncated non-signaling variant and the chemokines is affected by the immediate expression of HSV Control of early gene promoters. In some embodiments, the two chemokines are the same. In some embodiments, the two chemokines are different. In some embodiments, the two tumor-associated/specific antigens comprise two identical or different tumor-associated antigens. In some embodiments, the two tumor-associated/specific antigens comprise two identical or different tumor-specific antigens. In some embodiments, the two tumor-associated/specific antigens comprise one tumor-associated antigen and one tumor-specific antigen.

Accordingly, in some embodiments of the present disclosure, a truncated non-signaling non-signaling antigen encoding at least one tumor-associated/specific antigen is incorporated into the genome of the genetically modified oHSV. A first polynucleotide of the variant and a second polynucleotide encoding at least one chemokine, wherein the expression of the truncated non-signaling variant and the at least one chemokine is affected by the immediate expression of the HSV Control of early gene promoters.

In some embodiments, the genome of the genetically modified oHSV incorporates in the genome a first polynucleotide encoding a truncated non-signaling variant of a first tumor-associated/specific antigen, encoding a second tumor-associated A second polynucleotide of a truncated non-signaling variant of a specific antigen, and a third polynucleotide encoding a chemokine, wherein the truncated non-signaling variant and the The expression of the above chemokines is under the control of the immediate early gene promoter of HSV.

In some embodiments, the tumor-associated/specific antigen, the first or the second tumor-associated/specific antigen is independently selected from HER2, PSMA, BCMA, CD20, CD33, CD19, CD22, CD123, CD30, GPC- 3. CEA, claudin 18.2, EpCAM, GD2, MSLN, EGFR, MUC1, EGFRVIII, CD38, Trop-2, c-MET, Nectin-4, CD79b, CCK4, GPA33, HLA-A2, CLEC12A, p-calcium Mucin, TDO2, MART-1, Pmel 17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA 125, MG7-Ag, NY-CO-1 , RCAS 1, SDCCAG16, TAAL6 and TAG72 group. In some embodiments, the tumor-associated/specific antigen is selected from the group consisting of HER2, Trop-2, BCMA, and CD19.

In some embodiments, the chemokine is selected from the group consisting of CXCL1 to CXCL17, CCL1 to CCL28, XCL1, XCL2, and CX3CL1. In a preferred embodiment, the chemokine is selected from the group consisting of CXCL9, CXCL10, CXCL11, CXCL12, CCL3, CCL4, CCL5, CCL19, CCL21. In a preferred embodiment, tend to The chemokine is CCL5.

In some embodiments, the HSV immediate early gene promoter is the HSV-1 or HSV-2 immediate early gene promoter. In some embodiments, the immediate early gene promoter of HSV is selected from IE 1 (ICP0 promoter), IE 2 (ICP27 promoter), IE 3 (ICP4 promoter) and IE 4/5 (ICP22 and ICP47 promoter) group. In a preferred embodiment, the HSV immediate early gene promoter is the HSV-1 immediate early gene promoter IE4/5.

In some embodiments, the truncated non-signaling variant is the extracellular-transmembrane domain of a tumor-associated/specific antigen. For example, a truncated non-signaling variant of CD19 is the extracellular-transmembrane domain of CD19. For example, a truncated non-signaling variant of BCMA is the extracellular-transmembrane domain of BCMA. For example, a truncated non-signaling variant of HER2 is the extracellular-transmembrane domain of HER2. For example, a truncated non-signaling variant of Trop-2 is the extracellular-transmembrane domain of Trop-2. In some embodiments, the truncated non-signaling variant is the extracellular domain of a tumor-associated/specific antigen. In some embodiments, the truncated non-signaling variant is the extracellular domain linked to part of the transmembrane domain of the tumor-associated/specific antigen. In some embodiments, a truncated non-signaling variant is a variant of a wild-type tumor-associated/specific antigen that lacks part or all of the signaling domain.

In preferred embodiments, the genetically modified oHSV is derived from HSV type 1 (HSV-1) or HSV type 2 (HSV-2). In preferred embodiments, the genetically modified oHSV is derived from the F strain of HSV-1.

In a preferred embodiment, the polynucleotides described herein encode (i) CD19 truncated non-signaling variants of (ii) CCL5. In preferred embodiments, the polynucleotides encode (i) a truncated non-signaling variant of Trop-2, and (ii) CCL5. In preferred embodiments, the polynucleotides encode (i) a truncated non-signaling variant of HER2, and (ii) CCL5. In preferred embodiments, the polynucleotides encode (i) a truncated non-signaling variant of BCMA, and (ii) CCL5.

In preferred embodiments, the polynucleotides described herein encode (i) truncated non-signaling variants of CD19, (ii) truncated non-signaling variants of BCMA, and (iii) CCL5. In preferred embodiments, the polynucleotides described herein encode (i) truncated non-signaling variants of CD19, (ii) truncated non-signaling variants of Trop-2, and ( iii) CCL5. In preferred embodiments, the polynucleotides described herein encode (i) truncated non-signaling variants of CD19, (ii) truncated non-signaling variants of HER2, and (iii) CCL5.

In some embodiments, the tumor cells are solid tumor cells. In some embodiments, the tumor cells do not express tumor-associated antigens or tumor-specific antigens encoded by the polynucleotides. In some embodiments, the tumor cells express a tumor-associated or tumor-specific antigen encoded by a polynucleotide.

In some embodiments, a genetically modified oHSV as described above is further modified to delete segments of the nucleic acid of the genome of the oHSV, thereby attenuating the oHSV or removing certain characteristics that are not required for its intended use. In one embodiment, the genetically modified oHSV is deleted from an internal inverted repeat region, a segment encoding a viral gene, or both. In one embodiment, the segment of the nucleic acid of the deleted oHSV is the P prototype genome of the F strain of HSV-1 in locations 117005 to 132096. In one embodiment, the segment encoding a viral gene is a segment of a nucleic acid encoding γ34.5. In one embodiment, both copies of the gene γ34.5 are deleted.

In some embodiments, a genetically modified oHSV as described above is further modified to encode an immunostimulatory agent, an immunotherapeutic agent, or both. In one embodiment, the immunostimulant is selected from the group consisting of GM-CSF, IL-2, IL-12, IL-15, IL-24 and IL-27. In one embodiment, the immunotherapeutic agent is an anti-PD-1 antibody, an anti-CTLA4 antibody, or an antigen-binding fragment thereof. In one embodiment, the genetically modified oHSV encodes IL-12. In one embodiment, the genetically modified oHSV encodes an anti-PD-1 antibody or antigen-binding fragment thereof. In one embodiment, the genetically modified oHSV encodes IL-12 and an anti-PD-1 antibody or antigen-binding fragment thereof.

It should be noted that truncated non-signaling variants of tumor-associated/specific antigens and chemokine expression are required to be under the control of HSV immediate early gene promoters (such as the IE4/5 promoter) such that tumor-associated /Specific antigens are expressed shortly after viral infection and before viral replication leads to tumor cell lysis. The polynucleotide encoding the truncated non-signaling variant and the polynucleotide encoding the chemokine can be operably linked to the same immediate early promoter. In another embodiment, the polynucleotide encoding the truncated non-signaling variant and the polynucleotide encoding the chemokine may be operably linked to different immediate early promoters. When oHSV is further armed with immunostimulants, immunotherapeutics, or both, such as IL-12 and anti-PD-1 antibodies, the expression of immunostimulatory and/or immunotherapeutic agents is not necessarily under the control of the immediate early promoter, but Preferably regulated by different and relatively late promoters (eg CMV promoter or Egr-1 promoter) control. In one embodiment, the polynucleotide encoding IL-12 is operably linked to the Egr-1 promoter. In another embodiment, the polynucleotide encoding scFv-anti-hPD1 is operably linked to a CMV promoter.

In one embodiment, a first polynucleotide encoding a truncated non-signaling CD19 and a second polynucleotide encoding CCL5 are incorporated into the genome of the genetically modified oHSV, wherein the truncated non-signaling Expression of signaling CD19 and the CCL5 is under the control of the HSV-1 IE4/5 promoter.

In one embodiment, a first polynucleotide encoding a truncated non-signaling BCMA and a second polynucleotide encoding CCL5 are incorporated into the genome of the genetically modified oHSV, wherein the truncated non-signaling Expression of BCMA signaling and the CCL5 is under the control of the HSV-1 IE4/5 promoter.

In one embodiment, a first polynucleotide encoding a truncated non-signaling Trop-2 and a second polynucleotide encoding CCL5 are incorporated into the genome of the genetically modified oHSV, wherein the truncated The expression of the non-signaling Trop-2 and the CCL5 is under the control of the HSV-1 IE4/5 promoter.

In one embodiment, a first polynucleotide encoding a truncated non-signaling HER2 and a second polynucleotide encoding CCL5 are incorporated into the genome of the genetically modified oHSV, wherein the truncated non-signaling Expression of HER2 signaling and the CCL5 are under the control of the HSV-1 IE4/5 promoter.

In one embodiment, the genome of the genetically modified oHSV incorporates the coded A first polynucleotide encoding truncated non-signaling CD19, a second polynucleotide encoding truncated non-signaling BCMA9 and a third polynucleotide encoding CCL5, wherein the truncated The expression of non-signaling CD19, the truncated non-signaling BCMA9 and the CCL5 is under the control of the HSV-1 IE4/5 promoter.

In one embodiment, the genome of the genetically modified oHSV incorporates a first polynucleotide encoding truncated non-signaling CD19, a second polynucleotide encoding truncated non-signaling Trop-2 Acid and the third polynucleotide of coding CCL5, wherein the non-signaling CD19 of the truncated, the non-signaling Trop-2 of the truncated and the performance of the CCL5 are initiated by HSV-1 IE4/5 child control.

In one embodiment, a first polynucleotide encoding a truncated non-signaling CD19, a second polynucleotide encoding a truncated non-signaling HER2, and A third polynucleotide encoding CCL5, wherein the expression of said truncated non-signaling CD19, said truncated non-signaling HER2 and said CCL5 is controlled by the HSV-1 IE4/5 promoter.

In one embodiment, the genome of the genetically modified oHSV incorporates a first polynucleotide encoding a truncated non-signaling variant selected from any one of CD19, BCMA, Trop-2, and HER2, A second polynucleotide encoding CCL5, a third polynucleotide encoding an anti-PD-1 antibody, and a fourth polynucleotide encoding IL-12, wherein the truncated non-signaling CD19 and the The expression of CCL5 is controlled by the HSV-1 IE4/5 promoter, and the internal inverted repeat region of oHSV is deleted.

In one embodiment, the genome of the genetically modified oHSV incorporates the coded A first polynucleotide encoding a truncated non-signaling CD19, a second polynucleotide encoding a truncated non-signaling variant selected from any one of BCMA, Trop-2 and HER2, encoding A third polynucleotide of CCL5, a fourth polynucleotide encoding an anti-PD-1 antibody, and a fifth polynucleotide encoding IL-12, wherein the truncated non-signaling CD19 and the CCL5 The expression of is subject to the control of HSV-1 IE4/5 promoter.

In one embodiment, the genome of the genetically modified oHSV incorporates a first polynucleotide encoding a truncated non-signaling CD19 encoding a truncated gene selected from any one of BCMA, Trop-2, and HER2. A second polynucleotide encoding a non-signaling variant of the , a third polynucleotide encoding CCL5, a fourth polynucleotide encoding an anti-PD-1 antibody, and a fifth polynucleotide encoding IL-12 , wherein the expression of the truncated non-signaling CD19 and the CCL5 is under the control of the HSV-1 IE4/5 promoter, and wherein the internal inverted repeat region of the oHSV is deleted.

In one embodiment, the genome of the genetically modified oHSV incorporates a first polynucleotide encoding a truncated non-signaling CD19 encoding a truncated gene selected from any one of BCMA, Trop-2, and HER2. A second polynucleotide encoding a non-signaling variant of the , a third polynucleotide encoding CCL5, a fourth polynucleotide encoding an anti-PD-1 antibody, and a fifth polynucleotide encoding IL-12 , wherein the expression of the truncated non-signaling CD19 and the CCL5 is under the control of the HSV-1 IE4/5 promoter, and wherein the internal inverted repeat region of the oHSV is deleted, and wherein all of the oHSV Single copy genes are preserved.

In one embodiment, the genome of the genetically modified oHSV incorporates a first polynucleotide encoding a truncated non-signaling CD19 encoding a truncated gene selected from any one of BCMA, Trop-2, and HER2. The second polynucleotide encoding the non-signaling variant of A third polynucleotide of CCL5, a fourth polynucleotide encoding an anti-PD-1 antibody, and a fifth polynucleotide encoding IL-12, wherein the truncated non-signaling CD19 and the CCL5 The expression of the HSV-1 IE4/5 promoter is controlled by the internal inverted repeat region of the oHSV and two copies of γ34.5 are deleted, and all single-copy genes of the oHSV are retained.

In one embodiment, the PolyA tail is located downstream of the polynucleotide encoding the truncated antigen and chemokine. For example, polynucleotides encoding truncated non-signaling variants and chemokines are arranged 5'-CD19-CCL5-PolyA-3', 5'-BCMA-CCL5-PolyA-3', 5'-HER2 -CCL5-PolyA-3', 5'-CD19-BCMA-CCL5-PolyA-3', 5'-CD19-Trop-2-CCL5-PolyA-3' or 5'-CD19-HER2-CCL5-PolyA-3 '.

In one embodiment, incorporation of any of the polynucleotides encoding truncated non-signaling variants, immunotherapeutics and chemokines into the oHSV genome does not disrupt the function of the viral genes. For example, polynucleotides encoding anti-PD-1 antibodies or antigen-binding fragments thereof were introduced into the virus between the UL3 and UL4 genes, and polynucleotides encoding truncated non-signaling variants and chemokines were introduced into the virus Between the UL37 and UL38 genes. Furthermore, in one embodiment, the polynucleotide encoding IL2 replaces the internal inverted repeat region of the viral genome.

In the present disclosure, tumor-associated/specific antigens encoded by oHSV may be heterologous or homologous to oHSV-infected tumor cells. In one embodiment, the tumor cells express a tumor-associated/specific antigen that is different from the tumor-associated/specific antigen encoded by oHSV. For example, tumor cells overexpress CD22, whereas the genetically modified oHSVs of the disclosure express CD19, HER2, or both. In another embodiment, the tumor cells express the same tumor associated/specific antigen as encoded by oHSV. For example, tumor cell cells express HER2 to a low extent, whereas the genetically modified oHSVs of the present disclosure express HER2. In another embodiment, the tumor cells are free of known tumor-associated/specific antigens.

The genetically modified oHSV-infected tumor cells of the present disclosure are hematological tumor cells or solid tumor cells.

Advantageously, the presentation of a non-signaling tumor-associated/specific antigen on the surface of a tumor cell converts the tumor cell from a negative cell to a positive cell for that particular tumor-associated/specific antigen, thereby targeting the tumor to a specific antigen. Response to therapy with tumor-associated/specific antigens or tumor-specific antigens. The expression of heterologous polynucleotides is under the control of immediate early gene promoters (eg, IE4/5 of HSV-1), so that translation products are produced at the very early stage of oHSV entry into tumor cells and replication. For example, oHSV can be modified so that it expresses a truncated non-signaling CD19, a transmembrane protein expressed specifically on normal and most tumor B cells. The truncated non-signaling CD19 is then presented on the tumor cell surface prior to cell lysis by oHSV infection and serves as a target for CD19-directed CAR-T therapies such as ^Kymriah® , ^Yescarta® , or ^Tecartus® . That is, the expression of non-signaling CD19 converts tumor cells normally insensitive to CD19-directed CAR-T therapy into tumor cells sensitive to CD19-directed CAR-T due to the lack of CD19 antigen on tumor cells. In this case, the tumor would respond to CD19-directed CAR-T therapy.

A key advantage of genetically modified oHSV encoding more than one non-signaling tumor-associated/specific antigen is that it provides a versatile tool for use in combination with different tumor antigen-targeted therapies without the need for separate oHSV is repeatedly designed, tested, and manufactured for a tumor antigen-targeted therapy. The results showed that when two different non-signaling tumor-associated When /specific antigens are encoded by the same oHSV, they can be successfully expressed and presented on the surface of tumor cells simultaneously before tumor cells are lysed by virus infection. The presentation of two or more different non-signaling tumor-associated/specific antigens will convert tumor cells into double or even triple positive tumor cells, thereby making different tumor-targeted therapies effective against tumor cells. This will help improve the specificity and efficacy of corresponding tumor-targeted therapies, such as CAR-T cell therapy.

A further advantage of some of the genetically modified oHSVs disclosed herein is that, in addition to tumor-associated/specific antigens, they encode at least one chemokine, the expression and release of which further facilitates the trafficking of immune cells to tumor cells and infiltration. It is therefore particularly advantageous when CAR-T, CAR-NK, etc. are used in combination with the oHSV described herein. However, without being bound by any theory, the genetically modified oHSV disclosed herein can be used alone, and the secretion of chemokines will induce the chemotaxis of the body's immune cells (such as T cells) to tumors, and together with the anti-tumor effect of the virus Kill tumor cells.

Combination of oHSV and tumor-targeted therapy

Another aspect of the present disclosure pertains to combinations of any of the genetically modified oHSVs described above with tumor-targeting therapeutics for the treatment of various cancers. As noted above, the genetically modified oHSVs disclosed herein express non-signaling tumor-associated/specific antigens, which are then presented on the surface of tumor cells. This provides an opportunity for therapeutics designed to target tumor-associated/specific antigens to target oHSV-infected tumor cells.

In the present disclosure, the tumor-targeting therapeutic has a targeting moiety specific for a truncated non-signaling variant of at least one tumor-associated/specific antigen encoded by oHSV fraction, and effector moieties for killing or inhibiting the proliferation of cancer cells. When oHSV enters and replicates in tumor cells, the targeting moiety is specific for non-signaling tumor-associated/specific antigens presented on the surface of tumor cells. For example, the targeting moiety is the antigen binding domain of an antibody directed against a tumor-associated/specific antigen, such as the chimeric antigen receptor portion of an antibody, scFv, Fab, or CAR-T cell. Effector moieties can be used to kill cancer cells or inhibit the proliferation of cancer cells. For example, the effector moiety is an immune cell, including T cells and natural killer cells, a portion of a BiTE that can engage a T cell, or the drug portion of an antibody-drug conjugate.

In some embodiments, the tumor-targeted therapeutic is selected from chimeric antigen receptor T (CAR-T) cells, chimeric antigen receptor NK (CAR-NK) cells, bispecific T cell engagers (BiTEs), and Antibody Drug Conjugates (ADCs). In some embodiments, the tumor-targeted therapeutic is a CAR-T cell. In some embodiments, the tumor-targeted therapeutic is a CAR-NK cell. In some embodiments, the tumor-targeting therapeutic is a BiTE. In some embodiments, the tumor-targeted therapeutic is an ADC.

In some embodiments, the tumor-targeting therapeutic is a CAR-T cell targeting CD19. In some embodiments, the tumor-targeting therapeutic is a BiTE targeting CD19 or EpCAM. In some embodiments, the tumor-targeting therapeutic is an ADC targeting HER2, Trop-2, Nectin-4, BCMA, CD33, CD30, CD22, or CD79b.

In some embodiments, the tumor-targeting therapeutic is a CAR-T cell targeting CD19. In some embodiments, the tumor-targeted therapeutic agent is selected from the group consisting of Tecartus®, ^Kymriah® , ^Yescarta® , ^JWCAR -029, IM19CAR-T, CNCT19, BZ019, HD CD19 CAR-T, pCAR-19B, CD19-CART, CT032, iPD1 CD19 eCAR-T, LCAR-B38M, CT103A, CAR-BCMA T, AU-101, 4SCAR-PSMA, PSMA-CART, P-PSMA-101, C-CAR066, MB-CART20.1, PBCAR20A, LB1095, LB1901 , PRGN-3006, AMG553, CT041, CD30.CAR-T and CAR-GPC3 T group.

In some embodiments, the tumor-targeting therapeutic is a BiTE targeting CD19 or EpCAM. In some embodiments, the tumor-targeting therapeutic is selected from the group consisting of ^Blincyto® , AMG420, PF-3135, and GBR1302.

In some embodiments, the tumor-targeting therapeutic is an ADC targeting HER2, Trop-2, Nectin-4, BCMA, CD33, CD30, CD22, or CD79b. In some embodiments, the tumor targeting therapeutic is selected from the group consisting of Kadcyla ^® , Enhertu ^® , SHR-A1811, TAA013, RC-48, BAT8001, ARX788, A166, Trodelvy ^® , BAT8003, DAC-002, DS-1062, SKB264, Group consisting of RC-108, TR1801-ADC, Padccv ^® , Polivy ^® , Adcetris ^® , Mylotarg ^® , Blenrep ^® , PSMA ADC, ADCT-402, PTK7-ADC, and TRS005.

In a preferred embodiment, the genetically modified oHSV for use in combination with any of the tumor-targeting therapeutics described above is a genetically modified oHSV having a polynucleotide incorporated into the genome of said oHSV, said polynucleotide Encoding (a) truncated non-signaling variants of two tumor-associated/specific antigens and (b) chemokines, wherein expression of the truncated non-signaling variants and chemokines is controlled by Control of the HSV immediate early gene promoter. In some embodiments, the two tumor-associated/specific antigens comprise two identical or different tumor-associated antigens. In some embodiments, the two tumor-associated/specific antigens comprise two identical or Different tumor-specific antigens. In some embodiments, the two tumor-associated/specific antigens comprise one tumor-associated antigen and one tumor-specific antigen.

In preferred embodiments, the genetically modified oHSV for use in combination with a tumor-targeting therapeutic is a genetically modified oHSV expressing truncated non-signaling variants of CD19 and BCMA and CCL5, and The tumor-targeted therapeutic agent is a CAR-T cell targeting CD19 (such as Kymriah ^® , Yescarta ^® or Tecartus ^® ), a BiTE targeting CD19 (such as Blinatumomab), an ADC targeting BCMA (such as Blenrep ^® ), or any combination thereof.

In preferred embodiments, the genetically modified oHSV for use in combination with a tumor-targeting therapeutic is a genetically modified oHSV expressing truncated non-signaling variants of CD19 and HER2 and CCL5, and The tumor-targeting therapeutic agent is CD19-targeting CAR-T cells (such as Kymriah ^® , Yescarta ^® or Tecartus ^® ), CD19-targeting BiTEs (such as Blinatumomab), HER2-targeting ADCs (such as Kadcyla ^® or Enhertu ^® ), or any combination thereof.

In preferred embodiments, the genetically modified oHSV for use in combination with a tumor targeting therapeutic is a genetically modified oHSV expressing truncated non-signaling variants of CD19 and Trop-2 and CCL5 , and the tumor-targeting therapeutic agent is CAR-T cells targeting CD19 (such as Kymriah ^® , Yescarta ^® or Tecartus ^® ), BiTEs targeting CD19 (such as Blinatumomab), ADCs targeting Trop-2 (such as Trodelvy ^® ), or any combination thereof.

The combination of oHSV and tumor-targeted therapy can be embodied, for example, as a medical kit. because Here, in one aspect, there is provided a medical kit for treating cancer, which respectively comprises a genetically modified oncolytic herpes simplex virus (oHSV) as described herein and a tumor-targeting therapeutic agent, wherein the The tumor-targeted therapeutic agent has a targeting moiety specific for a truncated non-signaling variant of at least one tumor-associated/specific antigen encoded by a polynucleotide and is used for killing or inhibiting cancer cell proliferation the effect part.

In some embodiments, a kit of medicine for treating cancer comprises, respectively: a genetically modified oncolytic herpes simplex virus (oHSV) encoding (i) a truncated non-signaling variant of CD19, (ii) truncated non-signaling variants of BCMA, and (iii) CCL5; and CAR-T, ADC or BiTE targeting CD19 or BCMA. In some embodiments, the CAR-T, ADC or BiTE targeting CD19 or BCMA is selected from ^Tecartus® , ^Kymriah® , ^Yescarta® , ADCT-402 (ADC Therapeutics), Blinatumomab, JNJ-68284528 (JNJ-4528, Legend Biotech ), Blenrep (or GSK2857916), AMG420 (Amgen) and PF-3135 (Pfizer).

In some embodiments, a kit of medicine for treating cancer comprises, respectively: a genetically modified oncolytic herpes simplex virus (oHSV) encoding (i) a truncated non-signaling variant of CD19, (ii) truncated non-signaling variants of Trop-2, and (iii) CCL5; and CAR-Ts, ADCs or BiTEs targeting CD19 or Trop-2. In some embodiments, the CAR-T, ADC or BiTE targeting CD19 or Trop-2 is selected from the group consisting of ^Tecartus® , ^Kymriah® , ^Yescarta® , ADCT-402 (ADC Therapeutics), Blinatumomab, and ^Trodelvy® (Immunomedics) group.

In some embodiments, a kit of medicine for treating cancer comprises, respectively: a genetically modified oncolytic herpes simplex virus (oHSV) encoding (i) a truncated non-signaling variant of CD19, (ii) truncated non-signaling variants of HER2, and (iii) CCL5; and CAR-Ts, ADCs or BiTEs targeting CD19 or HER2. In some embodiments, the CAR-T, ADC or BiTE targeting CD19 or HER2 is selected from ^Tecartus® , ^Kymriah® , ^Yescarta® , ADCT-402 (ADC Therapeutics), Blinatumomab, AU-101 (Aurora Biopharma), ^Kadcyla® (Roche), ^Enhertu® (Daiichi Sankyo) and GBR1302 (Ichnos Sciences SA).

treatment method

Another aspect of the present disclosure relates to a method for treating cancer in a subject. The method comprises administering to the subject a therapeutically effective amount of a genetically modified oHSV as described herein and a tumor targeting therapeutic as described herein. Administration of oHSV and tumor-targeted therapeutics is performed simultaneously or sequentially.

In some embodiments, the subject is first administered a therapeutically effective amount of a genetically modified oHSV as described herein, followed by a tumor-targeting therapeutic as described herein. In this embodiment, the interval between administrations is in the range of 0.5-12 hours, such as 0.5-9 hours, 0.5-8 hours, 0.5-7 hours, 0.5-6 hours, 0.5-5 hours, 0.5-4 hours , 0.5-3 hours, 0.5-2 hours, 0.5-2.5 hours, 0.5-1.5 hours or 0.5-1 hours. For example, the administration of oHSV is 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, or 12 hours.

In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a genetically modified oncolytic herpes simplex virus (oHSV) encoding: (i) a truncated non-signaling variant of CD19 (ii) truncated non-signaling variants of BCMA, and (iii) CCL5; and CAR-Ts, ADCs or BiTEs targeting CD19 or BCMA. In some embodiments, the CAR-T, ADC or BiTE targeting CD19 or BCMA is selected from ^Tecartus® , ^Kymriah® , ^Yescarta® , ADCT-402 (ADC Therapeutics), Blinatumomab, JNJ-68284528 (JNJ-4528, Legend Biotech ), Blenrep (or GSK2857916), AMG420 (Amgen) and PF-3135 (Pfizer).

In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a genetically modified oncolytic herpes simplex virus (oHSV) encoding: (i) a truncated non-signaling variant of CD19 body, (ii) a truncated non-signaling variant of Trop-2, and (iii) CCL5; and a CAR-T, ADC or BiTE targeting CD19 or Trop-2. In some embodiments, the CAR-T, ADC or BiTE targeting CD19 or Trop-2 is selected from the group consisting of ^Tecartus® , ^Kymriah® , ^Yescarta® , ADCT-402 (ADC Therapeutics), Blinatumomab, and ^Trodelvy® (Immunomedics) group.

In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a genetically modified oncolytic herpes simplex virus (oHSV) encoding: (i) a truncated non-signaling variant of CD19 (ii) truncated non-signaling variants of HER2, and (iii) CCL5; and CAR-Ts, ADCs or BiTEs targeting CD19 or HER2. In some embodiments, the CAR-T, ADC or BiTE targeting CD19 or HER2 is selected from ^Tecartus® , ^Kymriah® , ^Yescarta® , ADCT-402 (ADC Therapeutics), Blinatumomab, AU-101 (Aurora Biopharma), ^Kadcyla® (Roche), ^Enhertu® (Daiichi Sankyo) and GBR1302 (Ichnos Sciences SA).

The combined use of oHSV described herein with various tumor antigen-directed CAR-T cells, ADCs, or BiTEs provided significantly enhanced antitumor effects against various tumors. oHSV directly disrupts the barrier and manipulates the tumor microenvironment through direct tumor cell lysis. Armed with payloads such as chemokines and cytokines, oHSV further improved T cell trafficking and infiltration into the tumor mass. In addition, the highly tumor-specific antigens (e.g., CD19, BCMA) delivered by oHSV on the surface of solid tumor cells also improve tumor-targeted therapies (e.g., CAR-T therapy) by reducing on-target off-tumor toxicity. ) specificity and safety.

sequence

The amino acid or nucleic acid sequences described in this disclosure are provided in Table 1 below.

Table 1 Amino acid or nucleic acid sequences described in this disclosure

A first aspect of the present disclosure relates to a genetically modified oncolytic herpes simplex virus (oHSV), wherein a polynucleotide is incorporated into the genome of said oHSV, said polynucleotide encoding (a) at least one tumor-associated The truncated non-signaling variant of / specific antigen and (b) at least one chemokine, wherein the expression of the truncated non-signaling variant and at least one chemokine is affected by the immediate early gene of HSV Control of a promoter, and wherein when said oHSV replicates in a tumor cell, said truncated non-signaling variant is expressed and presented on the surface of the tumor cell as a biomarker, and at least one chemokine is expressed and release to induce chemotaxis of immune cells towards tumor cells.

Another aspect of the present disclosure relates to a genetically modified oncolytic herpes simplex virus (oHSV), wherein a polynucleotide encoding at least one tumor-associated/specificity is incorporated into the genome of the oHSV The truncated non-signaling variant of the antigen, wherein the expression of the truncated non-signaling variant is controlled by the immediate early gene promoter of HSV, and when the oHSV replicates in tumor cells, the The truncated non-signaling variants were expressed and displayed on the surface of tumor cells as biomarkers.

Another aspect of the present disclosure relates to a medical kit for treating cancer, comprising separately: a genetically modified oncolytic herpes simplex virus (oHSV) as described herein and A tumor-targeted therapeutic agent, wherein the tumor-targeted therapeutic agent has a targeting moiety specific for a truncated non-signaling variant of at least one tumor-associated/specific antigen encoded by a polynucleotide and is used for The effector part for killing or inhibiting the proliferation of cancer cells.

Another aspect of the present disclosure relates to a method for treating cancer in a subject, comprising simultaneously or sequentially administering a pharmaceutically effective amount of a genetically modified oncolytic herpes simplex virus (oHSV) and a tumor-targeting therapeutic agent to said subject, wherein a polynucleotide is incorporated into the genome of said oHSV, said polynucleotide encoding (a) a truncated non-signaling variant of at least one tumor-associated/specific antigen, and preferably (b) at least one chemokine, wherein the truncated non-signaling variant and preferably at least one chemokine expression is under the control of the immediate early gene promoter of HSV, and wherein the tumor targeting The therapeutic agent has a targeting moiety specific for a truncated non-signaling variant of at least one tumor-associated/specific antigen encoded by the polynucleotide and is effective for killing or inhibiting the proliferation of cancer cells part.

Other aspects of the present disclosure will be readily apparent from the following detailed description described with reference to the accompanying drawings.

Figure 1 shows a schematic diagram of the oHSV backbone of T7201, T7202, T7203, T7204, T7011, T7012 and T7013 (hereinafter collectively referred to as "T7 series"). (A) Schematic representation of T3011, a genetically modified oHSV that encodes hPD-1 antibody (hPD-1-Ab), an immune checkpoint inhibitor, and hIL-12, a cytokine, whose internal The repetitive region (b'a' a'c') was replaced by a polynucleotide encoding hIL-12, and the expression cassette of hPD-1-Ab was introduced between the genes UL3 and UL4 of the UL fragment. A more detailed description of T3011 can be found in WO 2017/181420 (IMMV503), the entire contents of which are incorporated herein by reference. (B) Schematic representation of exemplary T7-series viruses described herein. These genetically modified oHSVs encode hPD-1-Ab, hIL-12, tumor-associated antigens, and chemokines, and their internal inverted repeats (b'a' and a'c') are multinucleated by hIL-12 Instead of nucleotides, the expression cassette of hPD-1-Ab was introduced between the genes UL3 and UL4 of the UL fragment, and the TAA+ chemokine expression cassette was introduced between the genes UL37 and UL38 of the UL fragment. (C) Schematic representation of exemplary genetically modified oHSV T7201, T7202, T7203, and T7204, wherein the TAA+ chemokine expression cassette in panel B is represented as a truncated non- signaling variants) and a chemokine. (D) Schematic representation of exemplary genetically modified oHSV T7011, T7012, and T7013, where the TAA+chemokine expression cassette in panel B is specifically two different TAAs plus one chemokine, expressed as TAA1+TAA2+chemokine . The HSV-IE (immediate early) promoter and PolyA tail were located upstream and downstream of the expression cassette, respectively.

Figure 2 shows a flowchart of the construction of the T7 series of oHSVs (ie T7011 to T7013 and T7201 to T7204 as described above). Construction involves multiple steps of cloning by means of the bacterial artificial chromosome (BAC) system.

Figure 3 shows the release of CCL5 after T7011 infection of 293T, HEp-2 and Tca8113 cells. The expression and release of CCL5 is rapid and intense. Secreted CCL5 was detected 4 hours after infection with a peak value as high as 5,000 pg/ml. After T7011 virus infection, secretions were stable and maintained for at least 4 days. Thus indicating CCL5 secretion.

Figure 4 shows the expression of truncated CD19, BCMA, Trop-2, HER2 on the cell surface. Different truncated antigens encoded by T7011, T7012 and T7013 were simultaneously expressed in tumor on the surface of tumor cells.

Figure 5 shows the anti-tumor effect of T7 series (T7011, T7012 and T7013) oHSV viruses. The _IC50 values of T7 series oHSV viruses are comparable to T3011, indicating that T7 series viruses have similar broad-spectrum antitumor activity compared with T3011.

Figure 6 shows that T7 series (T7011, T7012 and T7013) oHSV viruses have no infectious activity in CAR-T cells or normal T cells.

Figure 7 shows that T7 series (T7011, T7012 and T7013) oHSV viruses have no cell killing activity in CAR-T ^CD19 cells or normal T cells.

Figure 8 shows that the anti-tumor effect was significantly enhanced in the combination therapy of T7011 and CAR-T ^CD19 .

Figure 9 shows that T7011 virus infection can specifically cooperate with CAR-T ^CD19 anti-tumor activity.

Figure 10 shows that the anti-tumor effect was significantly enhanced in the combination therapy of T7012 and CAR-T ^CD19 .

Figure 11 shows that T7012 virus infection can specifically cooperate with CAR-T ^CD19 anti-tumor activity.

Figure 12 shows that the anti-tumor effect is significantly enhanced in the combination therapy of T7013 and CAR-T ^CD19 .

Figure 13 shows that T7013 virus infection can specifically cooperate with CAR-T ^CD19 anti-tumor activity.

Figure 14 shows that T7 series (T7011, T7012 and T7013) oHSV viruses have no cell killing ability in CAR-NK ^CD19 or NK cells.

Figure 15 shows viral replication of HSV-1(F) and T7011 in CAR-NK ^CD19 and NK cells.

Figure 16 shows that T7011 has no negative effect on the proliferation of CAR-NK ^CD19 cells. *p<0.05, ***p<0.001.

Figure 17 shows that T7011 has no negative effect on NK cell proliferation. *p<0.05, **p<0.01, ***p<0.001.

Figure 18 shows that the anti-tumor effect is significantly enhanced in the combination therapy of T7011 and CAR-NK ^CD19 .

Figure 19 shows that T7011 virus infection can specifically cooperate with CAR-NK ^CD19 anti-tumor activity.

Construction of oHSV-1 T7201, T7202, T7203, T7204, T7011, T7012 and T7013

Oncolytic herpes simplex virus (oHSV-1) T7201, T7202, T7203, and T7204 carry IL-12, anti-PD-1 antibody, CCL5, and a truncated nonspecific antigen (TAA) as a biomarker Coding sequences for signaling variants. T7201, T7202, Truncated non-signaling variants of the biomarkers expressed by T7203 and T7204 were CD19, BCMA, Trop-2 and HER2, respectively. Figure 1C shows a schematic diagram of the viral backbone of T7201 to T7204.

Oncolytic herpes simplex virus (oHSV-1) T7011, T7012, and T7013 carry two truncated non-signals of IL-12, anti-PD-1 antibody, CCL5, and tumor-associated antigen (TAA) as biomarkers Coding sequences of transduced variants. The truncated non-signaling variants of the biomarkers represented by T7011, T7012, and T7013 were CD19 plus BCMA, CD19 plus Trop-2, and CD19 plus HER2. Figure 1D shows a schematic representation of the viral backbones of T7011, T7012 and T7013. Figure 2 shows a flowchart of the construction of T7 series oHSVs.

The two biomarker and CCL5 coding sequences joined by the T2A self-cleaving peptide sequence (SEQ ID NO: 1) are translated in one open reading frame driven by the HSV-1 immediate early gene promoter (IE4/5 promoter). The expression cassette was inserted between the UL37 and UL38 genes.

In addition, T7201, T7202, T7203, T7204, T7011, T7012, and T7013 contain an insertion of an anti-human PD-1 antibody expression cassette between UL3 and UL4 , and a modified internal repeat (IR) region replaced by an IL-12 expression cassette . Recombinant viruses are constructed in several steps with the aid of the bacterial artificial chromosome (BAC) system. Details of virus construction are described below.

The flanks of the IL-12 expression cassette are respectively amplified from the HSV-1 viral genome by two sets of primers ((SEQ ID Nos: 2-3) and (SEQ ID Nos: 4-5)) under the content of the wild-type genome Upstream of nucleotide 117005 and downstream of nucleotide 132096, and insert the gene to replace pKO5 in plasmid pKO5 to generate pKO1407. pKO1407 was then transfected into E. coli with wild-type BAC by electroporation to generate BAC-T2010. Then, drive the anti-PD-1 antibody base The flanks of the CMV promoter box of the cause are respectively through two groups of primers ((SEQ ID Nos: 6-7) and (SEQ ID Nos: 8-9)) from HSV-1 viral genome PCR amplification under the content of wild-type genome Upstream of nucleotide 11658 and downstream of nucleotide 11659 were added, and ligated into pKO5 at the BglII and PacI sites to generate the pKOE1002 plasmid. The pKOE1002 plasmid was then transfected into E. coli containing BAC-T2010 by electroporation to generate BAC-T3011. Expression cassettes containing one or both biomarkers (ie, tumor-associated/specific antigens) and the CCL5 gene were flanked by upstream nucleotide 84220 and downstream nucleotide 84221 in the context of the wild-type genome. The upstream and downstream flanking sequences were amplified by PCR from the HSV-1 viral genome through two sets of primers ((SEQ ID Nos: 10-11) and (SEQ ID Nos: 12-13)), respectively. The DNA fragment contained the biomarker CCL5, and flanking sequences were ligated into pKO5 at XbaI and PacI sites to generate pKO7201, pKO7202, pKO7203, pKO7204, pKO7011, pKO7012 and pKO7013 plasmids. Then, pKO7201, pKO7202, pKO7203, pKO7204, pKO7011, pKO7012 and pKO7013 plasmids were transfected into E. coli containing BAC-T3011 by electroporation to produce BAC-T7201, BAC-T7202, BAC-T7203, BAC-T7204, BAC-T7011, BAC-T7012, BAC-T7013. T7201, T7202, T7203, T7204, T7011, T7012, and T7013 viruses were obtained by transfecting the corresponding BAC plasmids, followed by several steps of plaque purification and amplification in Vero cells.

Virus titration

Virus titers were measured by plaque formation assay. Briefly, virus stocks were serially diluted and then seeded as monolayers of Vero cells in T25 flasks. After 2 hours of adsorption, the culture medium was replaced with DMEM medium supplemented with 1% FBS plus 0.05% (wt/vol) human mixed immunoglobulin. Culture medium for 72 hours. Cells were fixed with anhydrous methanol for 5 min, rinsed with distilled water and stained with crystal violet. Lysed plaques were counted to calculate infectious viral particle titers. The titers of T7011, T7012 and T7013 are shown in Table 2 below, and the titers of T7201, T7202, T7203 and T7204 are shown in Table 4.

Table 2 T7011, T7012 and T7013 oHSV virus titers

Detection of CCL5 secretion after virus infection

Human embryonic kidney 293T, human laryngeal carcinoma Hep-2 and human tongue squamous carcinoma Tca8113 cells were inoculated into T25 flasks at a density of 1×10 ⁶ cells/flask. After overnight incubation, cells were either mock-infected or infected with 1 PFU/cell of T7011. After 2 hours of incubation, the inoculum was replaced with fresh medium. Cell supernatants were harvested at 0, 4, 8, 12, 24, 48, 72 and 96 hours post infection for ELISA assays to quantify CCL5 secretion. Figure 3 shows the release of CCL5 after T7011 infection in 293T, Hep-2 and Tca8113 cells. The expression and release of CCL5 is rapid and intense. Secreted CCL5 was detected 4 hours after infection with a peak value as high as 5,000 pg/ml. After T7011 virus infection, secretions were stable and maintained for at least 4 days. Thus indicating the secretion of CCL5.

ELISA to measure the expression of IL-12, anti-PD-1 antibody and CCL5

Seed Vero cells into T150 flasks at a density of ⁶ ×106 cells/flask. After overnight incubation, cells were infected with 0.01 PFU/cell of T7201, T7202, T7203, T7204, T7011, T7012 and T7013. Cell supernatants collected 48 hours after infection were used for ELISA assays to detect the expression levels of IL-12, anti-PD-1 antibody, and CCL5. The results are shown in Table 3 and Table 4. As shown in Table 3, the expression of CCL5 was at a high level and very stable among the tested viruses. The expression of IL-12 and PD-1 Ab was basically the same between T7011 and T7013, while T7012 had the highest expression of IL-12 and the lowest expression of PD-1 Ab.

Table 3 Expression degree of IL-12, anti-PD-1 antibody and CCL5 after virus infection

Table 4 T7201, T7202, T7203 and T7204 oHSV virus titers and expression levels of IL-12, anti-PD-1 Fab and CCL5 after virus infection

Expression and presentation of truncated CD19, BCMA, Trop-2, HER2 on the cell surface by immunofluorescence

Hep-2 cells (4×10 ⁵ ) were seeded on slides in individual wells of 6-well dishes and incubated for 24 hours to allow cell adhesion. Cells were then mock-infected or exposed to 5 PFU/cell of T7011, T7012 and T7013 virus for 1 hour, respectively. Replace the inoculum with fresh medium. Cells were rinsed with PBS and fixed with 4% paraformaldehyde for 10 min at the indicated times at room temperature, followed by blocking with 5% skim milk. T7011-infected cells were co-stained with CD19 (Cat.302204, Biolegend) antibody and BCMA (Cat.NBP1-97637, Novus) antibody; T7012-infected cells were stained with CD19 (Cat.302204, Biolegend) antibody and Trop-2 ( Cat.PA5-47030, Invitrogen) antibody co-staining; T7013-infected cells were stained with CD19 antibody (Cat.302204, Biolegend) and HER2 primary antibody (Cat.MAB1129-100, R&D systems) at 4°C overnight. Cells were then incubated with Alexa Fluor 488-conjugated anti-mouse (Cat.A32766, Invitrogen), Alexa Fluor 568-conjugated anti-rabbit (Cat.A11036, Invitrogen) and Alexa Fluor 568-conjugated anti-goat (Cat.A11057, Invitrogen) secondary antibody was incubated at room temperature for 1 hour. Cells were then washed with PBS and embedded in mountant (Cat. 8961S, Cell Signaling Technology). Images were captured and processed using a Nikon confocal laser scanning microscope (HD25, magnification, 120x), as shown in Figure 4. It can be seen from FIG. 4 that different truncated antigens encoded by T7011, T7012 and T7013 were simultaneously expressed on the surface of tumor cells.

Neurotoxicity Studies

Six-week-old female BALB/c mice were anesthetized, and then 50 μL of 10-fold serial dilutions of dilutions of HSV-1(F), T3011, T7011, T7012, or T7013 virus were injected intracranially to 8 mice per group. The same volume of DPBS containing 10% glycerol was inoculated as the mock treatment control group. The mice were monitored for 14 days, and the 50% lethal dose (LD50) was calculated from the mortality data according to Reed and Muench's method.

As shown in Table 5 below, the LD ₅₀ values of T7011, T7012, T7013 and T3011 are 158 times, 316 times, 100 times and 268 times higher than HSV-1(F) respectively, indicating that they are the same as T3011 virus and HSV-1(F) ), the neurotoxicity of T7011, T7012 and T7013 was significantly weakened.

Table 5 LD ₅₀ values of T7011, T7012, T7013, T3011

Antitumor Activity of T7 Series oHSV Viruses

Tumor cells were seeded on 96-well plates at a density of 10,000 cells/well. After overnight incubation, cells were infected with 0.01, 0.1, 1, 5, 10, 33.33 and 100 PFU/cell of T3011, T7011, T7012 and T7013 in triplicate. 48 hours post-infection (pi), cell viability was measured by CellTiter-Glo. Calculate the cell growth inhibition rate according to the manufacturer's instructions. The concentration ( _IC50 ) resulting in 50% inhibition of cell growth by virus infection (PFU/cell) was calculated by fitting the data to a dose-response curve using GraphPad Prism software.

As shown in Figure 5, the IC ₅₀ values of T7 series viruses are comparable to T3011, indicating that T7 series viruses have similar broad-spectrum anti-tumor activity compared with T3011. At the same time, the _IC50 values of HCT116, Hep-2, PC-3, MDA-MB-231 and A375 cells were slightly higher than those of other tumor cell lines, indicating that these cell lines were relatively resistant to infection by T7 series viruses and then selected for further combination studies.

Infectious Activity of T7 Series oHSV Viruses

Hep-2 cells, non-transduced normal T cells, and CD19 CAR-T (CAR-T) cells were seeded on 12-well plates at a density of 5×10 ⁵ cells/well, and 1 PFU/cell of HSV- 1(F), T7011, T7012 and T7013 infection. Cell pellets were harvested at 24 and 48 hours (h) after infection and washed with PBS. Cell pellets were then resuspended in DPBS+10% glycerol, followed by three freeze-thaw cycles. Viral progeny were titrated on Vero cells.

As shown in Figure 6, the virus yields in Hep-2 cells infected with all viruses were significantly higher than those in normal T cells and CAR-T cells. In particular, the production of T7 series viruses in normal T cells and CAR-T cells did not exceed 10 ³ PFU/mL at 24 hours or 48 hours after infection. These results indicated that wild-type HSV-1(F) virus had low infectious activity in CAR-T or normal T cells, while attenuated T7011, T7012, and T7013 viruses had no infectious activity.

Cell Killing Activity of T7 Series oHSV Viruses

CD19 CAR-T (CAR-T ^CD19 ) cells and non-transduced normal T cells were seeded on 96-well plates at 4×10 ⁴ cells/well, and T7011, 0.01, 0.1, 1, and 10 PFU/cell T7012 and T7013 infection, triplicate. Cell viability was measured by CellTiter-Glo at 24 and 48 hours post infection (pi). Relative cell viability was calculated as a percentage of untreated cells.

As shown in Figure 7, the cell viability did not decrease after T7 series virus infection, which proves that T7 series viruses have no cell killing activity on CAR-T or normal T cells.

T7011 and CAR-T ^CD19 Combination of antitumor effects

Human laryngeal cancer cells Hep-2, human melanoma cells A375 and human prostate cancer PC-3 cells were seeded on 96-well plates at a density of ¹ ×104 cells/well. After overnight incubation, cells were not infected or infected with 0.01, 0.03, 0.1, 0.3 and 1 PFU/cell of T7011 in triplicate. 24 hours after infection, 4×10 ⁴ cells/well of CAR-T ^CD19 or T cells were added at a ratio of 4:1 effector to target (E:T) for co-culture with tumor cells. Non-transduced normal T cells were used as controls. After 24 hours of co-culture, cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

60%的效果。結果表明，與單獨的T7011和CAR-T組相比，T7011和CAR-T^CD19組合治療組的抗腫瘤作用顯著增強。相比之下，T7011和正常T細胞組合治療顯示出輕微的抗腫瘤作用。 As shown in Figure 8, the combination of T7011 and CAR-T ^CD19 showed higher

60% effect. The results showed that the anti-tumor effect of the T7011 and CAR-T ^CD19 combination treatment group was significantly enhanced compared with the T7011 and CAR-T group alone. In contrast, combination therapy of T7011 and normal T cells showed a slight antitumor effect.

Further, human laryngeal cancer cell Hep-2, human melanoma cell A375 and human prostate cancer PC-3 cell were seeded on a 96-well plate at a density of 1×10 ⁴ cells/well. After overnight incubation, cells were infected with no or with 1 PFU/cell of T7011 or T3011 in triplicate. 24 hours after infection, 4×10 ⁴ cells/well of CAR-T ^CD19 cells were added at a 4:1 effector-to-target (E:T) ratio for co-culture for an additional 24 hours. Untreated cells were used as untreated controls. Cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

As shown in Figure 9, only T7011 and CAR-T ^CD19 combination treatment significantly reduced cell viability compared with the single treatment group and the T3011 and CAR-T combination treatment group. As a control, the combination of T3011 and CAR-T ^CD19 had no effect. All these results indicated that T7011 virus infection can specifically synergize the anti-tumor activity of CAR-T ^CD19 .

T7012 and CAR-T ^CD19 Combination of antitumor effects

Human laryngeal cancer cells Hep-2, human melanoma cells A375 and human prostate cancer PC-3 cells were seeded on 96-well plates at a density of ¹ ×104 cells/well. After overnight incubation, cells were infected without or with 0.01, 0.03, 0.1, 0.3 and 1 PFU/cell of T7012 in triplicate. 24 hours after infection, 4×10 ⁴ cells/well of CAR-T ^CD19 or T cells were added at a ratio of 4:1 effector to target (E:T) for co-culture with tumor cells. Non-transduced normal T cells were used as controls. After 24 hours of co-cultivation, cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

50%的效果。結果表明，與單獨的T7012和CAR-T組相比，T7012和CAR-T^CD19組合組的抗腫瘤作用顯著增強。相比之下，T7012和正常T細胞組合顯示出輕微的抗腫瘤作用。 As shown in Figure 10, the combination of T7012 and CAR-T ^CD19 showed higher

50% effect. The results showed that the anti-tumor effect of the T7012 and CAR-T ^CD19 combination group was significantly enhanced compared with the T7012 and CAR-T group alone. In contrast, the combination of T7012 and normal T cells showed a slight antitumor effect.

Further, human laryngeal cancer cell Hep-2, human melanoma cell A375 and human prostate cancer PC-3 cell were seeded on a 96-well plate at a density of 1×10 ⁴ cells/well. After overnight incubation, cells were either uninfected or infected with 1 PFU/cell of T7012 or T3011 in triplicate. 24 hours after infection, 4×10 ⁴ cells/well of CAR-T ^CD19 cells were added at a 4:1 effector-to-target (E:T) ratio for co-culture for an additional 24 hours. Untreated cells were used as untreated controls. Cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

As shown in Figure 11, only T7012 and CAR-T ^CD19 combination treatment significantly reduced cell viability compared to the monotherapy group and the T3011 and CAR-T combination treatment group. As a control, the combination of T3011 and CAR-T ^CD19 had no effect. All these results indicated that T7012 virus infection can specifically synergize the anti-tumor activity of CAR-T ^CD19 .

T7013 and CAR-T ^CD19 Combination of antitumor effects

Human laryngeal cancer cells Hep-2, human melanoma cells A375 and human prostate cancer PC-3 cells were seeded on 96-well plates at a density of ¹ ×104 cells/well. After overnight incubation, cells were not infected or infected with 0.01, 0.03, 0.1, 0.3 and 1 PFU/cell of T7013 in triplicate. 24 hours after infection, 4×10 ⁴ cells/well of CAR-T ^CD19 or T cells were added at a ratio of 4:1 effector to target (E:T) for co-culture with tumor cells. Non-transduced normal T cells were used as controls. After 24 hours of co-cultivation, cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

60%的效果。結果表明，與單獨的T7013和CAR-T組相比，T7013和CAR-T^CD19組合組的抗腫瘤作用顯著增強。相比之下，T7013和正常T細胞組合顯示出輕微的抗腫瘤作用。 As shown in Figure 12, the combination of T7013 and CAR-T ^CD19 showed higher

60% effect. The results showed that the anti-tumor effect of the T7013 and CAR-T ^CD19 combination group was significantly enhanced compared with the T7013 and CAR-T group alone. In contrast, the combination of T7013 and normal T cells showed a slight antitumor effect.

Further, human laryngeal cancer cells Hep-2, human melanoma cells A375 and human prostate cancer PC-3 cells were seeded on 96-well plates at a density of 1×10 ⁴ cells/well. After overnight incubation, cells were either uninfected or infected with 1 PFU/cell of T7013 or T3011 in triplicate. 24 hours after infection, 4×10 ⁴ cells/well of CAR-T ^CD19 cells were added at a 4:1 effector-to-target (E:T) ratio for co-cultivation for an additional 24 hours. Untreated cells were used as untreated controls. Cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

As shown in Figure 13, only T7013 and CAR-T ^CD19 combination treatment significantly reduced cell viability compared with the monotherapy group and the T3011 and CAR-T combination treatment group. As a control, the combination of T3011 and CAR-T ^CD19 had no effect. All these results indicated that T7013 virus infection can specifically synergize the anti-tumor activity of CAR-T ^CD19 .

T7 series (T7011, T7012 and T7013) oHSV viruses in CAR-NK ^CD19 Cell killing activity in cells and NK cells

NK cells are isolated from PBMCs according to methods known in the art. CD19 CAR-NK (CAR-NK ^CD19 ) cells and non-transduced normal NK cells were seeded on 96-well plates at 4×10 ⁴ cells/well, and HSV- 1(F), T7011, T7012 and T7013 infection, triplicate. Cell viability was measured by CellTiter-Glo at 24, 48 and 72 hours post infection (pi). Relative cell viability was calculated as a percentage of untreated cells.

As shown in Figure 14, the cell viability was not reduced after the T7 series virus infection, which proved that the T7 series virus had no cell killing activity on CAR-NK cells or normal NK cells.

HSV-1(F) and T7011 in CAR-NK ^CD19 Viral replication in cells and NK cells

CAR-NK ^CD19 cells and NK cells were seeded at a density of ⁵ × 105 cells/well in 12-well dishes and incubated at 1 PFU in the presence of IL-2 (+IL-2) or in the absence of IL-2 HSV-1(F) and T7011 infection of /cells. Cell pellets were harvested at 2, 24, 48 and 72 hours (h) post infection. Cell pellets were then washed with PBS, then resuspended in DPBS + 10% glycerol, and freeze-thawed 3 times. Viral progeny were titrated on Vero cells.

As shown in Figure 15, the T7011 virus has no infectious activity on CAR-NK ^CD19 cells or normal NK cells.

T7011 to CAR-NK ^CD19 Effects on Cell Proliferation

CAR-NK ^CD19 cells were seeded at a density of ⁵ × 105 cells/well in 12-well dishes and infected with or without 0.1 in the presence of IL-2 (+IL-2) or in the absence of IL-2 Or HSV-1(F) and T7011 at 1 PFU/cell. Cell pellets were harvested at 24, 48 and 72 hours (h) post-infection and stained with trypan blue to determine viable cells.

As shown in Figure 16, T7011 had no negative effect on the proliferation of CAR-NK ^CD19 cells.

Effect of T7011 on the proliferation of CAR-NK cells

NK cells were seeded at a density of ⁵ × 105 cells/well in 12-well plates and infected or not infected with 0.1 or 1 PFU/well in the presence of IL-2 (+IL-2) or in the absence of IL-2. Cells for HSV-1 (F) and T7011. Cell pellets were harvested at 24, 48 and 72 hours (h) post-infection and stained with trypan blue to determine viable cells.

As shown in Figure 17, T7011 had no negative effect on NK cell proliferation.

T7011 and CAR-NK ^CD19 combined cell killing

Human laryngeal cancer cells Hep-2, human melanoma cells A375 and human prostate cancer PC-3 cells were seeded on 96-well plates. After overnight incubation, cells were infected with 0.01, 0.1 and 1 PFU/cell of T7011 in triplicate. Twenty-four hours after infection, CAR-NK ^CD19 cells were added at a 2:1 effector-to-target (E:T) ratio for co-culture with tumor cells. After an additional 24 or 48 hours, cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

As shown in Figure 18, the combination of T7011 and CAR-NK ^CD19 showed a higher effect than single agent.

Further, human melanoma cell A375 cells were seeded on a 96-well plate at a density of 1×10 ⁴ cells/well. After overnight incubation, cells were infected with 1 PFU/cell of NK, CAR-NK ^CD19 or T7011 in triplicate. 24 hours after infection, CAR-NK ^CD19 cells or NK cells were added at a 2:1 effector-to-target (E:T) ratio for co-culture for an additional 24 hours. Cell viability was measured by CellTiter-Glo. Relative cell viability was calculated as a percentage of untreated cells.

As shown in Figure 19, T7011 and CAR-NK ^CD19 combination treatment significantly reduced cell viability compared to the single treatment group and the T7011 and NK combination treatment group. All these results indicated that T7011 virus infection can specifically synergize the anti-tumor activity of CAR-T ^CD19 .

Claims (29)

A genetically modified oncolytic herpes simplex virus (oHSV), wherein a polynucleotide encoding (a) a truncation of at least one tumor-associated/specific antigen is incorporated into the genome of said oHSV The non-signaling transduction variant of, and (b) at least one chemokine, wherein the expression of the non-signaling variant of said truncating and said at least one chemokine is controlled by the immediate early gene promoter of HSV , and wherein said truncated non-signaling variant is expressed and presented on the surface of said tumor cell as a biomarker when said oHSV replicates in a tumor cell, and said at least one chemokine is expressed and Released to induce chemotaxis of immune cells towards said tumor cells. The genetically modified oHSV as claimed in claim 1, wherein said at least one tumor-associated/specific antigen is selected from HER2, PSMA, BCMA, CD20, CD33, CD19, CD22, CD123, CD30, GPC-3, CEA , Claudin 18.2, EpCAM, GD2, MSLN, EGFR, MUC1, EGFRVIII, CD38, Trop-2, c-MET, Nectin-4, CD79b, CCK4, GPA33, HLA-A2, CLEC12A, p-cadherin, TDO2, MART-1, Pmel 17, MAGE-1, AFP, CA125, TRP-1, TRP-2, NY-ESO, PSA, CDK4, BCA225, CA 125, MG7-Ag, NY-CO-1, RCAS 1 , SDCCAG16, TAAL6 and TAG72 group. The genetically modified oHSV according to claim 1 or 2, wherein the at least one chemokine is selected from the group consisting of CXCL1 to CXCL17, CCL1 to CCL28, XCL1, XCL2 and CX3CL1. The genetically modified oHSV as described in any one of claims 1 to 3, wherein said Said truncated non-signaling variant is an extracellular domain, an extracellular-transmembrane domain or has at least 90% amino acid sequence identity with said extracellular domain or said extracellular-transmembrane domain its equivalent. The genetically modified oHSV as described in any one of claims 1 to 4, wherein the immediate early gene promoter of HSV is selected from IE 1 (ICP0 promoter), IE 2 (ICP27 promoter) of HSV-1 , IE 3 (ICP4 promoter) and IE 4/5 (ICP22 and ICP47 promoter) group. The genetically modified oHSV of any one of claims 1 to 5, wherein the polynucleotide encodes truncated non-signaling variants of two tumor-associated/specific antigens; and at least one chemotherapeutic factor. The genetically modified oHSV according to any one of claims 1 to 6, wherein said at least one chemokine comprises CCL5. The genetically modified oHSV according to any one of claims 1 to 7, wherein the polynucleotide is inserted between UL37 and UL38. The genetically modified oHSV as described in any one of claims 1 to 8, wherein said polynucleotide codes: (a) a truncated non-signaling variant of CD19, a truncated non-signaling variant of BCMA and CCL5; (b) a truncated non-signaling variant of CD19, a truncated non-signaling variant of Trop-2 and CCL5; (c) a truncated non-signaling variant of CD19, a truncated non-signaling variant of HER2 and CCL5; (d) a truncated non-signaling variant of CD19 and CCL5; (e) a truncated non-signaling variant of Trop-2 and CCL5; (f) a truncated non-signaling variant of BCMA and CCL5; or (g) A truncated non-signaling variant of HER2 and CCL5. The genetically modified oHSV as described in any one of claims 1 to 9, wherein the immediate early gene promoter of HSV is the immediate early gene promoter IE4/5 of HSV-1. The genetically modified oHSV according to any one of claims 1 to 10, wherein a PolyA tail is located downstream of the polynucleotides encoding truncated antigens and chemokines. The genetically modified oHSV according to any one of claims 1 to 11, wherein the tumor cells are solid tumor cells. The genetically modified oHSV according to any one of claims 1 to 12, wherein the tumor cells do not express the tumor-associated/specific antigen encoded by the polynucleotide. The genetically modified oHSV according to any one of claims 1 to 13, wherein the oHSV is further modified such that a fragment of the nucleic acid of the oHSV is deleted. The genetically modified oHSV according to any one of claims 1 to 14, wherein the fragment of the nucleic acid of the oHSV is the internal inverted repeat region of the oHSV, a fragment encoding a viral gene or both. The genetically modified oHSV according to claim 14, wherein the fragment of the nucleic acid of the oHSV is the position 117005 to 132096 in the P prototype genome of the F strain. The genetically modified oHSV according to any one of claims 1 to 16, wherein the oHSV is further modified to encode an immunostimulatory agent, an immunotherapeutic agent or both. The genetically modified oHSV according to claim 17, wherein the immunostimulator is selected from the group consisting of GM-CSF, IL-2, IL-12, IL-15, IL-24 and IL-27. The genetically modified oHSV according to claim 17 or 18, wherein the immunotherapeutic agent is an anti-PD-1 antibody, an anti-CTLA4 antibody, or an antigen-binding fragment thereof. The genetically modified oHSV according to any one of claims 17 to 19, wherein the immunostimulatory agent is IL-12 and the immunotherapeutic agent is an anti-PD-1 antibody or an antigen-binding fragment thereof. A medical kit for treating cancer, which respectively comprises: oHSV and a tumor-targeted therapeutic agent as described in any one of Claims 1 to 20, wherein the tumor-targeted therapeutic agent has a combination of the polynucleoside The acid-encoded truncated non-signaling variant of said at least one tumor-associated/specific antigen has a specific targeting moiety and an effector moiety for killing or inhibiting cancer cell proliferation. The medical kit according to claim 21, wherein the tumor-targeting therapeutic agent is selected from the group consisting of CAR-T cells, CAR-NK cells, BiTEs and ADCs. The medicine set as described in claim 22, wherein the tumor-targeting therapeutic agent is selected from CAR-T cells targeting CD19, CAR-NK cells targeting CD19, and CD19 or Group of BiTEs of EpCAM. The medical kit according to claim 22, wherein the tumor-targeting therapeutic agent is an ADC targeting HER2, Trop-2, Nectin-4, BCMA, CD33, CD30, CD22 or CD79b. The medicine set as described in claim 22, wherein the tumor targeting therapeutic agent is selected from Tecartus ^® , Kymriah ^® , Yescarta ^® , JWCAR-029, IM19CAR-T, CNCT19, BZ019, HD CD19 CAR-T, pCAR- 19B, CD19-CART, CT032, iPD1 CD19 eCAR-T, LCAR-B38M, CT103A, CAR-BCMA T, AU-101, 4SCAR-PSMA, PSMA-CART, P-PSMA-101, C-CAR066, MB-CART20 .1, a group consisting of PBCAR20A, LB1095, LB1901, PRGN-3006, AMG553, CT041, CD30.CAR-T and CAR-GPC3 T. The medical kit according to claim 22, wherein the tumor-targeting therapeutic agent is selected from the group consisting of Blincyto ^® , AMG420, PF-3135 and GBR1302. The medicine set as claimed in item 22, wherein the tumor-targeting therapeutic agent is selected from Kadcyla ^® , Enhertu ^® , SHR-A1811, TAA013, RC-48, BAT8001, ARX788, A166, Trodelvy ^® , BAT8003, DAC- 002, DS-1062, SKB264, RC-108, TR1801-ADC, Padccv ^® , Polivy ^® , Adcetris ^® , Mylotarg ^® , Blenrep ^® , PSMA ADC, ADCT-402, PTK7-ADC and TRS005 group. A genetically modified oncolytic herpes simplex virus (oHSV), wherein a polynucleotide is incorporated into the genome of said oHSV, said polynucleotide encoding at least one tumor-associated A truncated non-signaling variant of a sex/specific antigen, wherein the expression of the truncated non-signaling variant is controlled by the immediate early gene promoter of HSV, and wherein the oHSV is expressed in tumor cells Upon replication the truncated non-signaling variants are expressed and presented on the surface of the tumor cells as biomarkers. A medical kit for treating cancer, which separately comprises: oHSV as described in Claim 28 and a tumor-targeted therapeutic agent, wherein the tumor-targeted therapeutic agent has the encoding for the polynucleotide encoded by the polynucleotide The truncated non-signaling variant of at least one tumor-associated/specific antigen has a specific targeting moiety and an effector moiety for killing or inhibiting cancer cell proliferation.

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